This invention relates to a method of measuring an analyte in a liquid sample using a dry analytical element which does not contain reagents capable of reacting directly with the analyte to produce a measurable change.
It has been conducted from long ago to diagnose human diseases by analyzing blood, urine or the like.
As a method therefor, there is the wet analysis using a solution reagent. This method has a long history, and various detecting reagents have been developed for many items. Various analyzers have also been developed which range from compact apparatuses to big scale apparatuses. Samples used for the wet analysis are plasma, serum, urine and the like, but whole blood samples are in general not used as it is.
In the wet analysis, reagents can be divided into several groups by considering their stability during stock, and dissolved and mixed at the time of use. It is also possible to divide the addition of respective reagents into several steps.
Moreover, since it is possible co prepare a suitable amount of each reagent by dissolving according to the number of samples to be measured, the measuring cost per one sample is reduced. It is troublesome to automate the measurement by combining the treatment of many solutions. However, there is a history to develop clinical analytical apparatuses, and various automatic apparatuses having a good efficiency have already been developed and put to practical use in every field requiring big, middle, small treating capacity according to great social requirement.
Disadvantages of the wet analysis are in the preparation and supply of samples. Since this method is composed on the premise of the measurement of transmitted light through a transparent solution, whole blood samples cannot be used as the samples to be measured without pretreatment. That is, after drawing whole blood samples, they are centrifuged, and the plasma or serum which is the supernatant is put into a sample cup, or the centrifuge tube as it is set in a measuring apparatus instead of the sample cup. In addition to the complexity in the above operations, there is a further problem that it is necessary to keep a great amount of whole blood samples for separating a sufficient amount of plasma or serum without contamination of red blood cells.
In order to obtain 200 μl of a plasma sample by centrifuging, usually, 1.5 to 2 ml of whole blood is necessary. Even though centrifuging and aftertreatment are conducted carefully, a minimum amount of whole blood is estimated to be about 500 μl.
On the other hand, a necessary amount of a sample is about 10 μl per one analytical item for the measurement, and accordingly, it is only 100 μl for 10 items and 200 μl for 20 items. Nevertheless, 2 to 20 ml of blood is actually drawn in hospitals or the like, that is, 50 to 100 times as much as the necessary plasma amount is drawn. Every person, even a healthy person, suffers with pain mentally and physically by inserting the needle of a syringe into blood tube and drawing blood. Particularly, persons having thin tubes constitutionally and sick persons suffer with pain accompanied with drawing beyond imagination, and patients who are repeatedly drawn greatly desire to reduce the amount of blood drawn to a minimum.
Incidentally, in the blood drawing room of hospitals, medical practitioners and clinics, whole blood samples drawn and put into a tube or a vacuum syringe are transported as it is or in cooled conditions to a central assay room or an assay center. That is, each blood sample is centrifuged after transportation, and separated into solid components such as red blood cells and plasma or serum which is used as the analytical sample. During the transportation, there is a possibility that biochemical reactions affecting the analysis proceed by the coexistance of red blood cells, but countermeasures are taken only against the variation factors which are known to affect analytical results remarkably great, such as inhibition to glycolysis and anticoagulation.
By taking the above matters into consideration, it is preferred that centrifuging is conducted immediately after blood drawing, but this is usually not done. Because, analytical methods using blood serum as the sample have been established historically, and it is necessary for the separation of serum to complete coagulation by leaving at least for 30 minutes to 1 hour. Moreover, when blood is centrifuged after adding an anticoagulant, according to samples, fibrins occasionally deposit between the centrifugation and the measurement by an analyzer. The above matters are liable to induce troubles in the transferring system, such as pipetting syringe or tubes in the analyzer.
Accordingly, it is desirable to obtain serum samples by centrifuging within about 1 hour after drawing blood. However, although it is possible in the assay in hospitals, in the case of assay centers requiring a time for the transportation of samples, the time when centrifuging is conducted quite varies, and the centrifuging is often conducted after one day or more.
As an analytical method which has conquered the disadvantages accompanied with the preparation and supply of samples, many dry analytical elements (also called analytical film, multilayer test strip, or the like) have been developed wherein all reagents necessary for qualitative or quantitative analysis are incorporated into a test paper or an analytical element such as multilayer anlaytical film, and are sold.
The dry analytical elements have the following characteristics.    1) All reagents necessary for analysis are incorporated into an analytical element.    2) Reactions necessary for analysis occur merely by spotting a sample (usually, plasma, serum or urine, as to partial items, whole blood).
The dry analytical elements are roughly divided into 3 groups according co their utilization field.    Group 1: The object is in screening by practitioners and at homes, and the results can be judged qualitatively (+/−) or semiquantitatively (about 5 degrees) by visual inspection.    Group 2: Measuring place can be relatively freely selected by combining an analyzer characterized by compact size and simple operation. Used in emergency assay rooms, childhood wards, practitioners, small scale hospitals, etc.    Group 3: Used for measuring routine assays in hospitals or assay centers using a fully automatic analyzer.
The construction and content of the anlaytical elements are different according to the above classification.
Representative anlaytical elements belonging to Group 1 are urine test paper and blood sugar test paper. Analytical operations of these papers are simple, and analyzer is not necessary. However, the results are rough (such as normal or abnormal), and it is premised that, in the necessary case, sample is measured again by another analytical means capable of obtaining a quantitative result.
In the anlaytical elements according to this method, operations by a specialist, such as clinical assay engineer, medical doctor and nurse, are not premised, and commonly, it is composed so that urine or whole blood can be used as a sample without any pretreatment.
The object of the analytical elements belonging to Group 2 is in quantitative analysis, and quantitative measurement by an apparatus is premised. Operation is relatively simple, although it is not simpler than Group 1, a specialyst such as a clinical assay engineer is not premised. As to samples, analytical elements to which anyone of whole blood, plasma, serum or wrine is applicable have gradually been developed, but analytical items measurable by whole blood are still 10 and several items which are relatively restricted.
The samples applicable to fully automatic analyzers in Group 3 are, in general, limited to plasma, serum and urine, and whole blood cannot be used as the sample. However, measurable items have gradually been increased, and analytical elements have already been developed for measuring at least 40 items.
However, in the dry anlaytical elements, all reagents necessary for reactions must be incorporated into the analytical element; Nevertheless, the characterics of the reagents are different respectively according to analytical items, and as a result, there is a great problem that much labor, time and equipment cost are consumed by developing recipe and optimizing manufacturing conditions.
Besides, since all reagents are contained, it is necessary to keep sufficiently dry conditions for the stock of the analytical elements for a long period. For that purpose, usually, each analytical element is packed by a moisture proof package, and if necessary, a drying agent is put in the package.
Even when the dry package and cooling preservation are employed, shelf life is only 1 to 2 years which elevates the price of the dry analytical elements.